The present invention relates to the discovery that a specific pentamer peptide amide, which corresponds to a viral capsid sequence, can be used to inhibit viral infection, including human immunodeficiency virus (HIV) infection. More specifically, medicaments comprising said pentamer peptide amide and methods of using said compounds for the prevention and treatment of viral infection, such as HIV infection, are provided.
All viruses are composed of a protein shell surrounding a nucleic acid containing core. The protein shell directly surrounding the viral nucleic acid is called a capsid, whereas, the complete protein-nucleic acid complex having both the capsid and the nucleic acid is called a nucleocapsid. Arenaviruses, rotaviruses, orbiviruses, retroviruses (including lentiviruses), papillomaviruses, adenoviruses, herpesviruses, paramyxovirus, myxovirus, and hepadnaviruses all exhibit these general structural features. (Virology, Fields ed., third edition, Lippencott-Raven publishers, pp 1513, 1645,1778, 2047, 2113, 2221, and 2717 (1996)).
The capsid is composed of many subunits (capsomeres) and capsomeres are formed from several homo- or hetero-polymers of protein. The noncovalent bonds between capsomeres in a viral assembly are of the same sort that stabilize a folded protein domain. The interface between two subunits can look very much like a single domain, with amino acid side chains tightly packed against one another. A common feature to most of the virus structures analyzed is the way in which a polypeptide chain from one capsomere can extend under or over domains of neighboring capsomeres. These extended polypeptide arms intertwine with other polypeptide arms and help to stabilize the capsid by initiating hydrophobic interactions, hydrogen bonding, and salt bridges. Contacts between individual capsomeres, and for some viruses also contacts with core proteins, determine the overall capsid structure and if a number of identical capsomeres are involved, repeated contacts occur and the resulting structure is symmetrical. (Ld. at 62).
Some simple viruses form spontaneously from their dissociated components while others require enzyme-catalyzed modifications of the capsomeres to trigger assembly. Viral self assembly is driven by the stability of the interactions between protein subunits under conditions that favor association. More complex viruses are often constructed from subassemblies that have undergone self assembly processes. (Ld. at pp 62, 70, 1646 and 1888). Although the capsids of many viruses differ in protein composition, a general viral structural design has evolved characterized by polymerized capsomeres that, in turn, are composed of several homo- or hetero- polymers of protein.
HIV is the name given to a lentivirus that infects humans and that causes acquired immuno-deficiency syndrome (AIDS). The lentivirus isolates from humans are grouped into one of two types (HIV-1 and HIV-2) on the basis of serologic properties and sequence analysis of molecularly cloned viral genomes. Genetically distinct lentiviruses have been obtained from several non-human primate species including African green monkeys, sooty magabeys, mandrills, chimpanzees, and sykes. Collectively, the lentivirus isolates from non-human primates are called SIV. Sequence analysis reveals that the genomes of some SIV strains and HIV-1 and HIV-2 strains exhibit a high degree of homology. Further, electron microscopy reveals that the ultrastructure of HIV and SIV are similar in that both have virions about 110 nm in diameter with a cone-shaped nucleocapsid surrounded by a lipid bilayer membrane that contains envelope glycoprotein spikes. (Id. at pp. 1882-1883).
HIV is a complex retrovirus containing at least seven genes. The viral structural genes, designated gag, pot, and env, respectively code for the viral core proteins, reverse transcriptase, and the viral glycoproteins of the viral envelope. The remaining HIV genes are accessory genes involved in viral replication. The gag and env genes encode polyproteins, i.e., the proteins synthesized from each of these genes are post-translationally cleaved into several smaller proteins.
Although the overall shape of HIV and SIV virions is spherical, the nucleocapsid is asymmetrical having a long dimension of about 100 nm, a wide free end about 40-60 nm, and a narrow end about 20 nm in width. The nucleocapsid within each mature virion is composed of two molecules of the viral single-stranded RNA genome encapsulated by proteins proteolytically processed from the Gag precursor polypeptide. Cleavage of the gag gene polyprotein Pr55gag by a viral coded protease (PR) produces mature capsid proteins. These gag gene products are the matrix protein (p17), that is thought to be located between the nucleocapsid and the virion envelope; the major capsid protein (p24), that forms the capsid shell; and the nucleocapsid protein (p9), that binds to the viral RNA genome. This proteolytic processing in infected cells is linked to virion morphogenesis. (Id. at pp 1886-1887).
The major capsid protein p24 (also called CA) contains about 240 amino acids and exhibits a molecular weight of 24-27 kD. The protein p24 self-associates to form dimers and oligomeric complexes as large as dodecamers. Genetic studies with mutations in the HIV-1 gag polyprotein have identified several functional domains in the p24 protein including the C terminal half of the molecule and a major homology region (MHR) spanning 20 amino acids that is conserved in the p24 proteins of diverse retroviruses. These mutations appear to affect precursor nucleocapsid assembly. (Id. at pp 1888-1889).
Since the discovery of HIV-1 as the etiologic agent of AIDS, significant progress has been made in understanding the mechanisms by which the virus causes disease. While many diagnostic tests have been developed, progress in HIV vaccine therapy has been slow largely due to the heterogeneous nature of the virus and the lack of suitable animal models. (See, e.g., Martin, Nature, 345:572-573 (1990)).
A variety of pharmaceutical agents have been used in attempts to treat AIDS. Many, if not all, of these drugs, however, create serious side effects that greatly limit their usefulness as therapeutic agents. HIV reverse transcriptase is one drug target because of its crucial role in viral replication. Several nucleoside derivatives have been found to inhibit HIV reverse transcriptase including azidothymidine (AZT, zidovidine(copyright)). AZT causes serious side effects such that many patients cannot tolerate its administration. Other nucleoside analogs that inhibit HIV reverse transcriptase have been found to cause worse side effects than AZT. Another drug target is the HIV protease (PR) crucial to virus development. PR is an aspartic protease and can be inhibited by synthetic compounds. (Richards, FEBS Lett., 253:214-216 (1989)). Protease inhibitors inhibit the growth of HIV more effectively than reverse transcriptase inhibitors but prolonged therapy has been associated with metabolic diseases such as lipodystrophy, hyperlipidemia, and insulin resistance.
Additionally, HIV quickly develops resistance to nucleoside/nucleotide analogue reverse transcriptase inhibitors and protease inhibitors. This resistance can also spread between patients. Studies have shown, for example, that one tenth of the individuals recently infected by HIV already have developed resistance to AZT, probably because they were infected by a person that at the time of transmission carried a virus that was resistant to AZT.
It would be useful in the treatment and prevention of viral infections, including HIV and SIV, to have specific and selective therapeutic agents that cause few, if any, side effects.
The present invention relates to the discovery that a pentamer peptide amide ALGPG-NH2 (SEQ. ID. NO.: 1) inhibits viral infectivity, specifically HIV. An intact capsid structure is of vital importance for the infectivity of a virion. A way to disrupt assembly of capsid protein macromolecules, that for their infectivity are dependent on di-, tri-, tetra-, or polymerization, is to construct small molecules that affect such protein-protein interactions. It is contemplated that the pentamer peptide amide ALGPG-NH2 (SEQ. ID. NO.: 1) inhibits viral infectivity by disrupting the proper assembly of the viral capsid. Thus, aspects of the present invention relate to the pentamer peptide amide ALGPG-NH2 (SEQ. ID. NO.: 1) and its ability to disrupt viral capsid assembly.
It is believed that ALGPG-NH2 (SEQ. ID. NO.: 1) binds to a protein that is involved in capsomere organization and capsid assembly of HIV-1, HIV-2, and SIV and thereby inhibits and/or prevents proper capsid assembly and, thus, viral infection. This pentamer peptide amide and peptidomimetics resembling its structure (collectively referred to as xe2x80x9cpeptide agentsxe2x80x9d) are used in a monomeric or multimeric form. These peptide agents are suitable for therapeutic and prophylactic application in mammals, including man, suffering from viral infection, specifically HIV infection.
In one embodiment, a composition for inhibiting viral replication in host cells infected with a virus comprises an effective amount of a peptide amide that comprises, consists of, or consists essentially of ALGPG-NH2 (SEQ. ID. NO.: 1). In some embodiments, the compositions described above are joined to a support and in other embodiments, the compositions described above are incorporated into a pharmaceutical having a pharmaceutically acceptable carrier.
Methods of inhibiting viral replication in a host cell are also embodiments of the present invention. One approach, for example, involves administering to a cell an effective amount of a peptide amide comprising, consisting of, or consisting essentially of ALGPG-NH2 (SEQ. ID. NO.: 1). The method described above can be supplemented with an antiviral treatment selected from the group consisting of nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. The ALGPG-NH2 (SEQ. ID. NO.: 1) used in the method above can be joined to a support or can be administered in a pharmaceutical comprising a pharmaceutically acceptable carrier.
In another embodiment, a composition for inhibiting HIV replication in host cells comprises an effective amount of a peptide amide that comprises, consists of, or consists essentially of ALGPG-NH2 (SEQ. ID. NO.: 1). In some embodiments, the ALGPG-NH2 (SEQ. ID. NO.: 1) is joined to a support and in other embodiments, it is incorporated into a pharmaceutical comprising a pharmaceutically acceptable carrier.
In another method, an approach to inhibit HIV replication in host cells is provided, which involves administering to said cells a peptide amide that comprises, consists of, or consists essentially of an effective amount of ALGPG-NH2 (SEQ. ID. NO.: 1). This method can also be supplemented by an antiviral treatment selected from the group consisting of nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. Further, the ALGPG-NH2 (SEQ. ID. NO.: 1) used in this method can be joined to a support or can be administered in a pharmaceutical comprising a pharmaceutically acceptable carrier.
In another method, an approach for interrupting viral capsid assembly is provided. This approach involves contacting a cell with an effective amount of a peptide amide that comprises, consists of, or consists essentially of an effective amount of ALGPG-NH2 (SEQ. ID. NO.: 1). The pentamer peptide amide ALGPG-NH2 (SEQ. ID. NO.: 1) used in this method can also be joined to a support or incorporated in a pharmaceutical.
In still another method, an approach for interrupting HIV capsid assembly is provided. This approach also involves contacting a cell with an effective amount of a peptide amide that comprises, consists of, or consists essentially of an effective amount of ALGPG-NH2 (SEQ. ID. NO.: 1). The pentamer peptide amide ALGPG-NH2 (SEQ. ID. NO.: 1) used in this method can also be joined to a support or incorporated in a pharmaceutical.
Methods of making antiviral pharmaceuticals are also embodiments of the invention. By one approach, a peptide amide that comprises, consists of, or consists essentially of an effective amount of ALGPG-NH2 (SEQ. ID. NO.: 1) is provided and then said peptide amide is mixed with a pharmaceutically acceptable carrier or is otherwise formulated for human use. Preferred antiviral pharmaceuticals are formulated for oral administration, however, many other routes of administration are contemplated. The antiviral pharmaceutical embodied herein can also include known antiviral compounds, for example, nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors incorporated into the pharmaceutical.